Rhodopsin-mediated photoreception in cryptophyte flagellates - PubMed (original) (raw)

Rhodopsin-mediated photoreception in cryptophyte flagellates

Oleg A Sineshchekov et al. Biophys J. 2005 Dec.

Abstract

We show that phototaxis in cryptophytes is likely mediated by a two-rhodopsin-based photosensory mechanism similar to that recently demonstrated in the green alga Chlamydomonas reinhardtii, and for the first time, to our knowledge, report spectroscopic and charge movement properties of cryptophyte algal rhodopsins. The marine cryptophyte Guillardia theta exhibits positive phototaxis with maximum sensitivity at 450 nm and a secondary band above 500 nm. Variability of the relative sensitivities at these wavelengths and light-dependent inhibition of phototaxis in both bands by hydroxylamine suggest the involvement of two rhodopsin photoreceptors. In the related freshwater cryptophyte Cryptomonas sp. two photoreceptor currents similar to those mediated by the two sensory rhodopsins in green algae were recorded. Two cDNA sequences from G. theta and one from Cryptomonas encoding proteins homologous to type 1 opsins were identified. The photochemical reaction cycle of one Escherichia-coli-expressed rhodopsin from G. theta (GtR1) involves K-, M-, and O-like intermediates with relatively slow (approximately 80 ms) turnover time. GtR1 shows lack of light-driven proton pumping activity in E. coli cells, although carboxylated residues are at the positions of the Schiff base proton acceptor and donor as in proton pumping rhodopsins. The absorption spectrum, corresponding to the long-wavelength band of phototaxis sensitivity, makes this pigment a candidate for one of the G. theta sensory rhodopsins. A second rhodopsin from G. theta (GtR2) and the one from Cryptomonas have noncarboxylated residues at the donor position as in known sensory rhodopsins.

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Figures

FIGURE 1

FIGURE 1

(A) The time course of photoaccumulation of G. theta cells (solid lines) and control C. reinhardtii cells (dashed line) at the illuminated side of the cuvette in response to 500 nm actinic light. The arrows indicate switching on and off the actinic light. The relative light intensities were, from top to bottom, 100%, 26.5%, and 7.5% for G. theta, and 2% for C. reinhardtii. (B) Spectral sensitivity of G. theta phototaxis. Bars represent the reciprocal of equal response quantum requirement for photoaccumulation during 50 s, calculated on the basis of fluence-response curves; the solid line represents the absorption spectrum of purified _Gt_R1. (C) Decrease in the relative sensitivity to long-wavelength light during actinic light stimulation. The ratio of the amplitudes of photoaccumulation by 500 nm and 450 nm is plotted as a function of the duration of actinic illumination. (D) Light-dependent inhibition of phototaxis in G. theta by hydroxylamine (1 mM final concentration). The photoaccumulation during 100 s after the onset of the actinic light is plotted as a function of incubation time of the sample in darkness (solid symbols) or under 10 W m−2 white light (open symbols). Squares, 450 nm actinic light; circles, 500 nm actinic light.

FIGURE 2

FIGURE 2

Photoinduced electrical signals in Cryptomonas sp. S2: a freshwater relative of G. theta. (A) Kinetics of light-induced currents (solid line) and a two-exponential fit of the current decay (dash-dotted line). The sign of the photoelectric signal measured under the same conditions in the flagellate green alga C. reinhardtii (dashed line) was inverted for easier comparison with the signal in Cryptomonas. PRC, photoreceptor current; RR, regenerative response. (B) A series of photoelectric signals in Cryptomonas measured at different flash intensities. (Inset) Fluence dependence of the photoreceptor current and its fitting with a two-exponential function (solid line). F2/F1 and A2/A1 are the ratios of computer-generated estimates for saturation levels and maximal amplitudes, respectively, of the first and second components.

FIGURE 3

FIGURE 3

Alignment of helix C regions of primary sequences of opsins from cryptophyte algae (protein names in boldface) and other type 1 opsins. Amino acid residues forming the retinal-binding pocket are marked with an asterisk. Carboxylated amino acid residues corresponding to the proton acceptor (Asp85) and proton donor (Asp96) in _Hs_BR are shaded black. _Hs_BR, Halobacterium salinarum bacteriorhodopsin; GPR, green-absorbing proteorhodopsin; BPR, blue-absorbing proteorhodopsin; _Np_SRII, Natronomonas pharaonis sensory rhodopsin II.

FIGURE 4

FIGURE 4

(A) Transmembrane topology of _Gt_R1 as predicted by TMHMM server version 2.0 (50). Arrows indicate the beginning of the long (1) and truncated (2) versions of the expressed protein. (B) Western blot analysis of long (1) and truncated His6-tagged (2) G. theta opsins expressed in E. coli. Numbers at the right indicate molecular weight markers.

FIGURE 5

FIGURE 5

(A) Absorption specta of purified long (solid line) and truncated (dashed line) _Gt_R1. (B) Laser flash-induced absorbance changes at 580 nm in long and truncated versions of _Gt_R1 expressed in E. coli cells (solid lines) and fits of their decay with exponential functions (dashed lines). (C) Kinetics of laser flash-induced absorbance changes in purified long _Gt_R1 at characteristic wavelengths, indicated in the figure, and their multiexponential fitting (dashed lines). (D) Spectra of laser flash-induced absorbance changes in long _Gt_R1. Solid circles and open circles represent 1 − 0 ms and 1 − 50 _μ_s in purified protein, respectively; open diamonds represent 10 − 0 ms in _Gt_R1 in E. coli cells.

FIGURE 6

FIGURE 6

Light-induced charge movement in _Gt_R1 and BPR measured in suspension of E. coli cells. Positive signal corresponds to transfer of a positive charge to outside the cell.

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